Analogues of parathyroid hormone (1-34) that function as agonists of the parathyroid hormone receptor-1 and display modified activity profiles

ABSTRACT

Described are polypeptide analogs of parathyroid hormone (PTH) that include an unnatural amino acid substitution at positions 7 or 8 from the N-terminus of the polypeptide. Also described are pharmaceutical compositions useful for treating hypoparathyroidism that contain the analogs and methods of using the analogs to treat hypoparathyroidism.

FEDERAL FUNDING STATEMENT

This invention was made with government support awarded under GM056414awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND

Proper function of individual cells and entire organisms depends uponinformation transfer from the extracellular environment to thecytoplasm. Most signals must be transduced via proteins that spancellular membranes. Many receptor proteins do not act as simple, binary“toggle switches,” with only signaling-active and signaling-inactivestates. Rather, they behave in vivo as nuanced interpreters of molecularinformation. This behavior enables the transmission of diverse messagesbased on variations in agonist structure. “Biased agonism” is onewidely-studied manifestation of this complexity that has been documentedfor multiple G protein-coupled receptors (GPCRs).^(1,2) Signaltransduction via these GPCRs involves multiple intracellular partners,only some of which are G proteins. A natural agonist activates thesealternative signaling pathways in a given proportion, for a given celltype and environment. Other agonists are designated as biased relativeto this benchmark if they lead to a different balance of signalintensities among the available pathways.³ These differences in signaltransduction pattern can arise from subtle agonist-dependent variationsin receptor conformation.⁴

The biased agonism paradigm is not the only mechanism by which diversityin GPCR signaling can arise from variations in agonist-bound receptorconformation. The parathyroid hormone receptor-1 (PTHR-1), for example,has two distinct functional states. These are depicted schematically inFIG. 1. The RG functional state, shown to the right in FIG. 1, formswhen the intracellular portion contacts a given G protein (designatedGα_(ND) in FIG. 1). In contrast, the R⁰ functional state, shown to theleft in FIG. 1, forms independent of G protein association.^(5,6) Anagonist's affinity for the RG state is predicted to correlate withPTHR-1 activation potency, while an agonist's R⁰ affinity correlateswith the duration of some in vivo responses.^(7,8) Natural agonists forPTHR-1 include parathyroid hormone (PTH) and parathyroid hormone-relatedprotein (PTHrP), which display similar affinity for the RG state butdiffer in their affinity for the R⁰ state.⁷ This behavior cannot bedescribed as biased agonism because PTH and PTHrP seem to activate thesame intracellular signaling mechanisms,⁹ but there is a clearmechanistic parallel to the bias paradigm in that agonists withdifferent receptor-state selectivities cause different biologicaleffects.^(7,8,10)

The signaling duration of PTHR1 is highly ligand dependent. Thetransient PTHR1 activation by PTHrP is primarily confined to cellmembrane, which follows the classic GPCR signaling model. On the otherhand, PTHR1 activation by PTH is more prolonged. As noted above, such adistinction of signaling mode was initially explained by a“conformational selectivity” model, which involves two conformationalstates, G protein-dependent (RG) versus G protein-independent (R0). Itis proposed that ligands with higher R0 binding affinity have longerreceptor residence time and thereby produce more sustained signalingresponse via more cycles of R0-to-RG isomerization. In parallel, thefunctional diversity of β-arrestin (“βarr”) in GPCR signaling hasreceived increasing investigation in recent years. Different from itsclassic role as signaling desensitizer, βarr was also found to act asthe scaffold protein to assemble the signaling complex. In the contextof PTHR1 signaling, it was discovered that sustained cAMP activation canoriginate from the endosome after internalization of theligand/GPCR/βarr ternary complex. In such a signaling mode, βarr plays akey role causing receptor internalization and assembling the downstreamsignaling cascade. PTHR1 agonists with high R0-binding affinity, such asPTH and LA-PTH, appear to favor the formation of the ternary complex.

Receptor state-selective agonists are highly prized because thesemolecules can serve as powerful tools for elucidatingsignal-transduction mechanisms, and they may give rise to therapeuticagents with minimal deleterious side effects.^(1,2) At present, there isno way to design such agonists via rational methods.

In terms of mammalian disease states, including humans, the umbrellaterm hypoparathyroidism is used to designate any decreased function ofthe parathyroid glands with concomitant underproduction of PTH. Thisthen leads to low levels of calcium in the blood. The main symptoms ofhypoparathyroidism are the result of the low blood calcium level, whichinterferes with normal muscle contraction and nerve conduction. As aresult, people with hypoparathyroidism experience a number of unsettlingsymptoms, including paresthesia (an unpleasant tingling sensation aroundthe mouth and in the hands and feet), muscle cramps, and tetany (severespasms that affect the hands and feet). Many subjects suffering fromhypoparathyroidism also report somewhat vague but pervasive symptomssuch as fatigue, headaches, bone pain and insomnia. Chronichypoparathyroidism is conventionally treated with vitamin D analogs andcalcium supplementation. However, such treatments are contra-indicatedin many patients due to potential renal damage. The N-terminal fragmentof parathyroid hormone, PTH (1-34), has full biological activity.Teriparatide (marketed in the U.S. by Eli Lilly & Co. under thetrademark “Forted”) is a recombinant form of PTH approved for use in thetreatment of osteoporosis.

SUMMARY OF THE INVENTION

G protein-coupled receptors (GPCRs), the targets of many currenttherapeutic agents, can adopt multiple activated states, and there isincreasing interest in synthetic molecules that display alteredreceptor-state selectivity patterns relative to natural agonists.Disclosed herein are backbone-modified analogs of a well-known peptideagonist, PTH(1-34). The PTH (1-34) analogues described herein are biasedtoward Gs activation/cAMP production relative to β arrestin recruitment.The analogs were generated via systematic replacement of selectedα-amino acid residues with either β-amino acid residues or withunnatural D-stereoisomer α-amino acid residues. Two distinct states ofPTHR-1 with high agonist affinity are known, and this system was used toassess the impact of backbone modification on binding preferences forthe alternative receptor conformations. The results show that biasedagonism can be achieved via this strategy. The resulting variations inagonist properties can give rise to distinct behaviors in vivo.

Thus, disclosed herein is a method to make unnatural PTHR-1 peptideagonists which exhibit biased agonism activity. The method comprisesdetermining or acquiring the α-amino acid sequence of a first PTHR-1peptide agonist that comprises α-amino acid residues, and thenfabricating an analog of the first PTHR-1 peptide agonist in which atleast one natural L-stereoisomer α-amino acid residue in position 6, 7,or 8 from the N-terminus of the PTHR-1 is replaced with a β-amino acidresidue or an unnatural D-stereoisomer α-amino acid residue. The naturalα-amino acid residue may optionally be replaced with a β-amino acid or aD-α-amino acid residue having the same side-chain as the natural α-aminoacid residue it replaces. Alternatively, at least one of the naturalα-amino acid residues may optionally be replaced with a cyclicallyconstrained β-amino acid residue. The same substitutions may also bemade at positions 1 and 2.

Also disclosed herein are the resulting unnatural, isolated peptideanalogs. Thus, disclosed herein are unnatural, isolated peptide analogscomprising PTH, a parathyroid hormone receptor (PTHR-1, PTHR-2) agonist-or antagonist- or inverse agonist effective fragment of PTH, aparathyroid hormone related protein (PTHrP), a PTHR-1 or PTHR-2agonist-, antagonist-, or inverse agonist-effective fragment of PTHrP,M-PTH, a PTHR-1 or PTHR-2 agonist-, antagonist-, or inverseagonist-effective fragment of M-PTH, BA058, or a PTHR-1 or PTHR-2agonist-, antagonist-, or inverse agonist-effective fragment of BA058,in which at least one natural L-stereoisomer α-amino acid residue inposition 6, 7, or 8 from the N-terminus of the protein or fragmentthereof is replaced with a β-amino acid residue or an unnaturalD-stereoisomer α-amino acid residue. As in the first embodiment, thesame substitutions may also be made at positions 1 and 2.

Salts of the foregoing peptide analogs are also within the scope of thisdisclosure.

In another version, the PTH analogs comprise thirty four (34) N-terminalresidues of a mammalian parathyroid hormone, PTH(1-34), in which atleast one natural L-stereoisomer α-amino acid residue in position 7 or 8from the N-terminus of the PTH(1-34) is replaced with a β-amino acidresidue or an unnatural D-stereoisomer α-amino acid residue.

Specific PTH analogs disclosed herein include:

(SEQ. ID. NO: 1) Ligand 1: SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF (SEQ. ID.NO: 2) Ligand 2: S VSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF (SEQ. ID. NO: 3)Ligand 3: S VSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF (SEQ. ID. NO: 4) Ligand 4:

VSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF (SEQ. ID. NO: 5) Ligand 5: S VSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF (SEQ. ID. NO: 6) Ligand 6: SVSE IQLMHNLGKHLNSMERVEWLRKKLQDVHNF (SEQ. ID. NO: 7) Ligand 7: SVSEI QLMHNLGKHLNSMERVEWLRKKLQDVHNF (SEQ. ID. NO: 8) Ligand 8:SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF (SEQ. ID. NO: 9) Ligand 9: SVSEIQ LMHNLGKHLNSMERVEWLRKKLQDVHNF (SEQ. ID. NO: 10) Ligand 10: SVSEIQ

MHNLGKHLNSMERVEWLRKKLQDVHNF (SEQ. ID. NO: 11) Ligand 11:SVSEIQL^(n)LHNLGKHLNSMERVEWLRKKLQDVHNF (SEQ. ID. NO: 12) Ligand 12:SVSEIQL^(n) LHNLGKHLNSMERVEWLRKKLQDVHNF (SEQ. ID. NO: 13) Ligand 13:SVSEIQL ^(n) L HNLGKHLNSMERVEWLRKKLQDVHNF (SEQ. ID. NO: 14) Ligand 14:SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF (SEQ. ID. NO: 15) Ligand 15: SVSEI QLMHNLGKHLNSMERVEWLRKKLQDVHNF (SEQ. ID. NO: 16) PTH (1-34)SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF bold = (R*) β² amino acid residuebold, underline = (S*) β² amino acid residue bold, double-underline= (S*) β³ amino acid residue bold, italics = (D)-α-amino acid residue nL= (R)-β²-Nle

The PTH analogs disclosed herein may be biased agonists of parathyroidhormone receptor-1.

Also disclosed herein are pharmaceutical compositions for treatinghypoparathyroidism. The composition comprises a parathyroid hormonereceptor agonist-effective amount of a compound or salt thereof asdisclosed herein in combination with a pharmaceutically suitablecarrier. Also disclosed herein is a method of treatinghypoparathyroidism in a mammalian subject, including a human subject.The method comprising administering to the subject a parathyroid hormonereceptor agonist-effective amount of a pharmaceutical composition asdescribed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of the G-protein uncoupled)(R⁰ and coupled(RG) conformational states of PTHR-1. “ECD” is extracellular domain.

FIG. 2 depicts graphs and a corresponding table reporting the EC50 andEmax of PTHR1 agonists PTH, LA-PTH, BA058 and PTHrP for cAMP signalingand β-arrestin (βarr) recruitment.

FIG. 3 is a histogram and corresponding table reporting the functionalselectivity of various PTHR1 agonists that display distinctly differentsignaling durations.

FIG. 4 discloses the sequences of exemplary ligands disclosed andclaimed herein and their respective activities to induce cAMP activity.

FIG. 5 depicts graphs and a corresponding table reporting the EC50 andEmax of PTHR1 agonists Ligands 8-13 (SEQ. ID. NOS: 8-13) for cAMPsignaling and β-arrestin (βarr) recruitment.

FIG. 6 is a histogram and corresponding table showing the bias factorsof Ligands 8, 10, and 12 (SEQ. ID. NOS: 8, 10, and 12).

FIG. 7 depicts two graphs reporting the results of washout assays onLigands 8, 10, and 12 (SEQ. ID. NOS: 8, 10, and 12).

FIG. 8 depicts a matched pair of graphs (“Ligand-on phase” vs.“Ligand-off phase”) reporting the effect of competitive antagonism onligand signaling duration.

FIG. 9 is a schematic diagram presenting a possible explanation for theobserved differences in signaling duration exhibited by differentligands.

FIG. 10A is a dose-response curve of PTHR1 activation in HEK293 cellsstably expressing PTHR1. Data represent mean±s.e.m from threeindependent measurements. Curves were fit to the data using afour-parameter sigmoidal dose-response equation.

FIG. 10B is a dose-response curve of PTHR2 activation in HEK293 cellsstably expressing PTHR2. For PTHR2 activation, data represent mean±s.e.mfrom four independent measurements. Binding data represent mean±s.e.mfrom three independent measurements. Curves were fit to the data using afour-parameter sigmoidal dose-response equation.

FIG. 11 is graph showing antagonistic effect of PTHrP(1-36)-NH₂ andα/β-peptide 14 on PTHR2 activation by PTH(1-34)-NH₂. Data pointsrepresent mean±s.e.m from four independent experiments. Curves were fitto the data using a four-parameter sigmoidal dose-response equation.

DETAILED DESCRIPTION Abbreviations and Definitions

Agonist, Antagonist, Inverse Agonist: An inverse agonist is an agentthat binds to the same receptor as an agonist but induces apharmacological response opposite to that agonist. A prerequisite for aninverse agonist response is that the receptor must have a constitutivelevel of activity in the absence of any ligand. An agonist increases theactivity of a receptor above its basal level, whereas an inverse agonistdecreases the activity below the basal level. An antagonist binds to thereceptor and blocks the activity of both agonists and inverse agonists.

“Cyclically constrained” when referring to a β-amino acid or β-aminoacid residue means a β-amino acid or β-amino acid residue in which theα-position and β-position carbon atoms in the backbone of the β-aminoacid are incorporated into a substituted or unsubstituted C₄ to C₁₀cycloalkyl, cycloalkenyl, or heterocycle moiety, wherein heterocyclemoieties may have 1, 2, or 3 heteroatoms selected from the groupconsisting of N, S, and O. Generally preferred cyclically constrainedβ-amino acids have the α-position and β-position carbon atoms in thebackbone incorporated into a substituted or unsubstituted C₅ to C₈cycloalkyl, cycloalkenyl, or heterocycle moiety having one or more N, S,or O atoms as the heteroatom. Within any given PTH analog, thecyclically constrained β-amino acid residues may be the same ordifferent.

DIEA=N,N-diisopropylethylamine.

DMF=dimethylformamide.

GPCR=G protein-coupled receptor.

HBTU=2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate.

HOBt=N-hydroxybenzotriazole.

MALDI-TOF-MS=matrix-assisted laser desorption/ionization time-of-flightmass spectrometry.

PTH=mammalian parathyroid hormone (including human PTH), its proprotein,its preproprotein, and any PTHR-1 agonist-effective fragment thereof. Inhumans, the corresponding PTH gene encodes and expresses a preproproteincomprising the amino acid sequence:

(SEQ. ID. NO: 20) MIPAKDMAKVMIVMLAICFLTKSDGKSVKKRSVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGE ADKADVNVLTKAKSQ.After post-translational processing, human PTH comprises the amino acidsequence

(SEQ. ID. NO: 21) SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKSQ.See U.S. National Center for Biotechnology Information (NCBI) referencesequence NP_000306 (online athttp://www.ncbi.nlm.gov/protein/NP_000306.1).

PTHR-1=parathyroid hormone receptor 1. hPTHR-1 designates the humanversion of the receptor; rPTHR-1 designates the rat version of thereceptor. The prefix or suffix WT in combination with either designates“wild-type.”

PTHrP=parathyroid hormone-related protein (SEQ. ID. NO: 19)

TFA=trifluoroacetic acid.

TBS=tris-buffered saline (i.e., tris(hydroxymethyl)aminomethane).

WT=wild-type.

Amino acid residues in the analogues disclosed herein that also appearin the corresponding natural protein/polypeptide are present in their Lconfiguration. Unnatural α-amino acid substitutions are present in theirD configuration. The terms “peptide” and “polypeptide” are usedsynonymously and refer to a polymer of amino acids which are linked viaamide linkages. β-amino acid residues may be linear, unsubstituted, orsubstituted at the α- or β-position carbon atoms of the backbone (i.e.,at the β² or β³ carbon atoms) or may be conformationally constrained bya cyclic group encompassing the α and β backbone carbon atoms of theinserted β-amino acid residue. While not required, it is preferred thatthe β-amino acid residues are corresponding β³ versions of the α-aminoacid residues they replace. That is, the side-chain on the β-positioncarbon (the β³ carbon) in the β-amino acid residue is the same as theside-chain on the α-amino acid residue it replaces and the α-positioncarbon (the β² carbon) in the β-amino acid residue is unsubstituted.

“Pharmaceutically suitable salts” means salts formed with acids or basesthe addition of which does not have undesirable effects whenadministered to mammals, including humans. Preferred are the salts withacids or bases listed in the U.S. Pharmacopoeia (or any other generallyrecognized pharmacopoeia) for use in humans. A host ofpharmaceutically-suitable salts are well known in the art. For basicactive ingredients, all acid addition salts are useful as sources of thefree base form even if the particular salt, per se, is desired only asan intermediate product as, for example, when the salt is formed onlyfor purposes of purification, and identification, or when it is used asintermediate in preparing a pharmaceutically-suitable salt by ionexchange procedures. Pharmaceutically-suitable salts include, withoutlimitation, those derived from mineral acids and organic acids,explicitly including hydrohalides, e.g., hydrochlorides andhydrobromides, sulphates, phosphates, nitrates, sulphamates, acetates,citrates, lactates, tartrates, malonates, oxalates, salicylates,propionates, succinates, fumarates, maleates, methylene bis bhydroxynaphthoates, gentisates, isethionates, di p toluoyltartrates,methane sulphonates, ethanesulphonates, benzenesulphonates, ptoluenesulphonates, cyclohexylsulphamates, quinates, and the like. Baseaddition salts include those derived from alkali or alkaline earth metalbases or conventional organic bases, such as triethylamine, pyridine,piperidine, morpholine, N methylmorpholine, and the like. Other suitablesalts are found in, for example, Handbook of Pharmaceutical Salts, P. H.Stahl and C. G. Wermuch, Eds., © 2011, Wiley-VCH (Zurich, Switzerland)ISBN: 978-3906390512, which is incorporated herein by reference.

Numerical ranges as used herein are intended to include every number andsubset of numbers contained within that range, whether specificallydisclosed or not. Further, these numerical ranges should be construed asproviding support for a claim directed to any number or subset ofnumbers in that range. For example, a disclosure of from 1 to 10 shouldbe construed as supporting a range of from 2 to 8, from 3 to 7, from 1to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.

All references to singular characteristics or limitations of the presentinvention shall include the corresponding plural characteristic orlimitation, and vice-versa, unless otherwise specified or clearlyimplied to the contrary by the context in which the reference is made.

All combinations of method or process steps as used herein can beperformed in any order, unless otherwise specified or clearly implied tothe contrary by the context in which the referenced combination is made.

The methods, compounds and compositions of the present invention cancomprise, consist of, or consist essentially of the essential elementsand limitations of the method described herein, as well as anyadditional or optional ingredients, components, or limitations describedherein or otherwise useful in synthetic organic chemistry, pharmacy,pharmacology, and the like.

Overview:

Alternative activated conformations of a receptor protein are likely todiffer from one another in subtle ways.⁴ Thus, making subtlemodifications to a natural agonist could be fruitful for makingcompounds with diverse selectivities among functional receptor states.In the present invention, an unconventional strategy was used in whichthe backbone of a natural PTHR-1 agonist was altered, rather than theside-chain complement. The results show that backbone-modification canrapidly identify potent agonists with divergent receptor stateselectivity patterns relative to a prototype peptide.

More specifically, the initial studies that yielded the compoundsdisclosed and claimed herein were conducted to determine whether thereis a correlation between βarr recruitment and receptor residence time ofa PTHR1 agonist. The initial studies were thus conducted in two steps.First, the functional selectivity (Gs activation as indicated by cAMPproduction vs. βarr recruitment) of several PTHR1 agonists that displaysubstantial differences in signaling duration were evaluated. Second,novel PTHR1 agonists were designed that have high functional selectivityand evaluated the signaling duration of these biased agonists.

Design of PTH(1-34) Analogs:

PTH is an 84-residue protein that controls key physiological processes,including the maintenance of extracellular levels of calcium andphosphate.⁹ The N-terminal fragment PTH(1-34) matches full-length PTH inpotency and efficacy at PTHR-1 and is the active ingredient in theosteoporosis drug teriparatide.¹¹ A crystal structure of the humanPTHR-1 extracellular domain (ECD) bound to PTH(15-34) reveals that thissegment forms an α-helix upon association with the receptor.¹² Thebioactive conformation of the N-terminal portion of PTH is unknown.PTH(1-34) analogs containing unnatural residue substitutions in theN-terminal region were fabricated and their in vitro and in vivoactivity as PTHR-1 agonists explored. Exemplary α/β-peptide analogsaccording to the present invention (working examples) are shown in FIG.4. Native α residues in the wild-type PTH were replaced with β³homologs. Thus, in the exemplary compounds the natural side chainsequence was maintained in the resulting α/β-peptides, but the backbonecontained additional CH₂ units.

Receptor Binding and Activation:

Well-established radio-ligand-displacement assays⁵⁻⁷ were used todetermine whether the α→β and L→D α replacements lead to variations inaffinities for the R⁰ or RG state of hPTHR-1 relative to the prototypeα-peptide PTH(1-34)^(14,15) Agonist activity was determined bymonitoring cAMP production in HEK293 cells that stably express PTHR-1and the GloSensor-brand cAMP reporter and PTHR-1.¹⁶

The results of the binding and activity assays support the hypothesisthat subtle modification of a prototype α-peptide via α→β and/or L→D αreplacements enables the discovery of agonists with variations inreceptor-state affinity profile relative to the α-peptide itself.

Functional Selectivity of Reported R0- and RG-Selective PTHR1 Agonists:

Relative to PTH (1-34) (SEQ. ID. NO: 16), BA058 (SEQ. ID. NO: 18; seeFIG. 2) and PTHrP (1-36) (SEQ. ID. NO: 19; see FIG. 2) display low andmoderate R0 affinity respectively, and these agonists induce signalingbias toward Gs/cAMP activation relative to PTH (1-34). The functionalselectivity of BA058 is more significant than that of PTHrP. Incontrast, LA-PTH (SEQ. ID. NO: 17; see FIG. 2), which has high R0affinity and induces prolonged endosomal signaling, appears to beslightly biased toward βarr recruitment. LA-PTH is a long-acting PTHanalog. See, for example, Shimuzu et al. (2016) “Pharmacodynamic Actionsof a Long-Acting PTH Analog (LA-PTH) in Thyroparathyroidectomized (TPTX)Rats and Normal Monkeys,” J Bone Miner Res. 31(7):1405-12. All fourligands show similar potency (EC50) in terms of βarr recruitment, butPTHrP and BA058 display a lower efficacy βarr recruitment maximum thando PTH and LA-PTH. See FIGS. 2 and 3. The top panel of FIG. 2 providesthe amino acid sequences for PTH(1-34), LA-PTH, BA058, and PTHrP. Themiddle panels in FIG. 2 are graphs depicting, from left-to-right: βarr1and βarr2 recruitment, presented as normalized BRET signals(bioluminescence resonance energy transfer) as a function of the log ofthe peptide concentration, and cAmp activation presented as normalizedRLU signals (relative light units) as a function of the log of thepeptide concentration. Red=PTH. Orange=LA=LA-PTH. Blue=BA058.Green=PTHrP. (BRET assays are well known and suitable kits are availablecommercially. See, for example, Promega's “NanoBRET” kits, catalogs nos.N1821 and N1811; Promega Corporation, Fitchburg, Wis.). The table at thebottom of FIG. 2 presents the pEC50 and Emax values for the βarr1 andβarr2 recruitment and cAMP activation. FIG. 3 presents the data for thebias of the activity—either toward βarr recruitment or cAMP activation.As seen in the histogram in FIG. 3, the activity of LA-PTH is biasedtoward βarr1 and βarr2 recruitment relative to cAMP activation. Incontrast, BA058 and PTHrP are both biased toward cAMP activationrelative to both βarr1 and βarr2 recruitment. The table in FIG. 3presents the bias factors for LA-PTH, BA058, and PTHrP.

Discovery of Highly G-Protein-Biased Signaling (“Gs”) PTHR1 Agonistswith Good Potency Via N-Terminal Modification of PTH:

In this work, it was found that several positions of N-terminal PTH cantolerate β² and/or β³ amino acid substitutions. New PTHR1 agonistshaving high functional selectivity and good ligand potency werefabricated as a result. See FIG. 4. The chart on the left of FIG. 4lists N-terminal amino acid positions on the Y-axis, and then indicateson the X-axis what types of amino acid substitutions at that amino acidposition are tolerated with respect to the ability of the resultingpeptide to activate cAMP production. The substitutions explores wereα→(R*)-β², α→(S*) β², α→(S*)-β³, (L)-α→(D)-α Other N-terminal sites werealso evaluated, but β substitutions at these sites generally led to adrop in potency and efficacy reduction for cAMP production and βarrestin recruitment.

Among Ligands 1-7 (SEQ. ID. NOS: 1-7, respectively), only Ligand 6,resulting from the incorporation of (S*)-β²Ile at the 5^(th) position ofPTH, induces appreciable functional selectivity toward Gs/cAMP; Ligands1-5 and 7 do not show significant signaling bias relative to PTH (1-34)(SEQ. ID. NO: 14). See Table 1.

In contrast, the 7^(th) and 8^(th) positions from the N-terminus ofPTH(1-34) (explored in Ligands 8-13; SEQ. ID. NOS: 8-13, respectively)are sensitive spots for high functional selectivity. See FIG. 5.Incorporating (R*)-β²Leu/(D)-Leu at the 7^(th) position or (R*)-β²Nle atthe 8^(th) position leads to high Gs-biased agonism. In contrast,(S*)-β²Leu at the 7^(th) position or (S*)-β²Nle/(L)-Nle at 8^(th)position does not induce significant signaling bias. (D)-Nle is poorlytolerated at the 8^(th) position for both cAMP signaling and βarrrecruitment. The top panels in FIG. 5 are graphs depicting, fromleft-to-right: βarr1 and βarr2 recruitment, presented as normalized BRETsignals as a function of the log of the peptide concentration, and cAmpactivation presented as normalized RLU signals as a function of the logof the peptide concentration. Red circles=PTH(1-34). Yellowsquares=Ligand 8. Blue upward triangles=Ligand 9. Green downwardtriangles=Ligand 10. Purple diamonds=Ligand 11. Green circles=Ligand 12.Black squares=Ligand 13. The table at the bottom of FIG. 5 presents thepEC50 and Emax values for the βarr1 and βarr2 recruitment and cAMPactivation.

As seen in the histogram in FIG. 6, the signaling activities of Ligands8, 10, and 12 are all strongly biased toward cAMP activation relative toboth βarr1 and βarr2 recruitment. The table in FIG. 6 presents the biasfactors for Ligands 8, 10, and 12.

Washout Assay of Gs-Biased PTHR1 Agonists:

Additional cell-based assays were pursued to elucidate the variationamong in vivo responses observed for Ligands 8, 10, and 12, ascontrasted to PTH(1-34). These “washout assays”^(7,8,21) assess theability of the peptides to form stable ligand-receptor complexes capableof stimulating prolonged cAMP responses. The ability of a peptide toelicit prolonged cAMP responses via PTHR-1 following washout typicallycorrelates with the affinity of that peptide for the R⁰ state.^(7,8,21)

The three highly Gs-biased PTHR1 agonists, Ligands 8, 10, and 12, weretested in the washout assay. See FIG. 7. Interestingly, they displayedvery different signaling durations. Both 8 and 12 induced more transientsignaling than PTH (1-34) (FIG. 7, left-hand panel), while the signalingduration of 10 was more prolonged than that of PTH (1-34) (FIG. 7,right-hand panel). These results suggest that the signaling duration ofa PTHR1 agonist cannot be predicted from its functional selectivity.

Utility of Highly Gs-Biased PTHR1 Agonists:

Gs-biased agonists of PTHR1 that cause transient receptor activation,like Ligands 8 and 12, useful for treating osteoporosis. Additionally,Gs-biased agonists that induce prolonged signaling duration, like Ligand10, are useful for treating hypoparathyroidism. Without being limited toany underlying mechanism or biologically phenomenon, the sustained cAMPactivation from a highly Gs-biased agonist, such as Ligand 10, couldresult from receptors that remain at the cell surface. This is becausethe relatively poor βarr recruitment ability of such a ligand shouldinhibit receptor internalization. (In contrast, sustained cAMPproduction induced by PTH (1-34) or LA-PTH involves internalizedreceptors). To test this hypothesis, a modified washout assay wasdesigned in which the signaling durations of several PTHR1 agonists werecompared in the presence of and in the absence of a non-internalizedcompetitive antagonist, (D)-Trp¹², Tyr³⁴-bPTH (7-34). The results ofthis washout assay are shown in FIG. 8. In the experiment, theantagonist ligand was introduced to the cells in the “ligand-off” phase.Compared to LA-PTH and PTH, Ligand 10 displayed a very striking changeof signaling duration upon the introduction of the competitiveantagonist. This strongly indicates that this biased agonist mainlystays at the cell surface and is highly defective in inducing PTHR1internalization.

Previous research shows that LA-PTH has a very short lifetime in thebloodstream despite its prolonged signaling effect; this behavior hasbeen explained by proposing that LA-PTH is very effective in inducinginternalization of the receptor-agonist complex and this internalizationremoves LA-PTH from the bloodstream. If this hypothesis is correct, thenit is possible that a highly Gs-biased agonist would have a longlifetime in the bloodstream, relative to LA-PTH, which might lead to animprovement in therapeutic effect for hypoparathyroidism.

Prolonged cAMP signaling of a PTHR1 agonist can cause both catabolic andanabolic effects. Intermittent PTH (1-34) administration is currentlyused to decouple bone resorption from bone formation. Spatial control ofcAMP production within the cell, resulting from GPCR endocytosis, can becrucial in terms of downstream effects of receptor activation. SeeIrannejad et al. (2017) “Functional selectivity of GPCR-directed drugaction through location bias,” Nature Chemical Biology 13:799-806. Thecatabolic and anabolic effects of PTHR1 agonism might therefore beinfluenced by how effectively an agonist induces receptor endocytosis.This phenomenon can be evaluated using the novel ligands describedherein. For example, despite its low efficiency in internalizing PTHR1,Ligand 10 can induce prolonged PTHR1 activation similar to PTH.Therefore, it is useful as a control ligand to study the potentialspatial effect of prolonged endosomal PTHR1 activation. FIG. 9 presentsa schematic representation of sustained endosomal signaling versus theproposed sustained signaling due to defective internalization.

The analogs are also useful as probes for pinpointing differences inligand recognition by PTH1R and PTH2R.

‘β Scan’ of the N-Terminal Portion of PTH(1-34)-NH₂.

Implementing backbone-modification involves replacing one α-amino acidresidue or more in an all-α sequence with a homologous β-amino acidresidue. The β residue retains the original side chain (or a similarside chain) but contains an additional methylene in the backbonerelative to the α residue that was replaced. β²-homoamino acids, inwhich the side chain projects from the carbon adjacent to carbonyl havereceived little attention in the scientific literature because ofdiminished accessibility. As shown here, though, including β²-homoaminoacids is important for achieving agonist selectivity.

B-family GPCRs feature a large extracellular domain that binds to theC-terminal portion of a peptide agonist such as PTH(1-34); this portionof the agonist is usually α-helical in the bound state. The N-terminalportion of the agonist engages the transmembrane domain of the GPCR, butthe receptor-bound conformations of most hormone N-terminal segments areunknown. In this work, it was hypothesized that α→β modifications in theN-terminal portion of PTH(1-34)-NH₂ would generate PTHR1 vs. PTHR2selectivity. Thus, a β-scan of the first eight residues of this agonistwas performed.

Many enantiopure β³-homoamino acids with protecting groups necessary forsolid-phase peptide synthesis are commercially available. In contrast,only a few protected β²-homoamino acids can be purchased. This practicaldistinction has skewed the functional evaluation of peptides containingβ-amino acid residues toward β³ residues. Most prior work on peptidesthat contain α→β. replacements (α/β-peptides) has focused on β³ residuesthat maintain the configuration of L-α-amino acids, which means S formost β³ residues but R in a few cases, such as β³-hSer or β³-hThr.Residues with this absolute stereochemistry, which we designate S* here[in other words, (R)-β³-hSer is designated (S*)-β³-hSer] can participatein right-handed α-helix-like secondary structures, as demonstratedcrystallographically for numerous α/β-peptides containing 25-33% βresidues distributed among L-α residues. In contrast, very little isknown about the conformational or biological properties of α/β-peptidescontaining β² residues. Here, a three-part approach to the N-terminalβ-scan of PTH(1-34)-NH₂ was undertaken in which each of the first eightresidues was replaced by the (S*)-β³, the (S*)-β² or the (R*)-β²homologue. For Met-8, the two β²-homonorleucine (β²-hNle) enantiomerswere employed.

Tables 2 and 3 summarize the effects of single α→β replacements on PTHR1and PTHR2 agonist activity.

Relative EC₅₀ values of PTH(1-34)-NH₂ analogues containing a singlehomologous α→β replacement within the first eight residues for PTHR1 andPTHR2 activation, as indicated by cAMP production, normalized to theEC⁵⁰ of PTH(1-34)-NH₂ itself. Green denotes tolerance of a α→β, i.e.,the corresponding α/β-peptide is a full PTHR1 agonist with EC50<2.5times the EC₅₀ of PTH(1-34)-NH₂. Yellow denotes tolerance of a α→βreplacement (analogous standard to that used for PTHR1). Striped boxdenotes poor tolerance of β amino acid for PTHR2 activationβ²-hNle was used instead of β²-hMet; both β³-hMet and β³-hNle weretested at the 8^(th) position; the value shown in table was the EC50 ofβ³-hMet (β³-hNle is poorly tolerated at the 8^(th) position for bothPTHR1 and PTHR2 activation)

The assays employ HEK-293 cells that have been engineered to express theappropriate receptor. GloSensor-based detection of cAMP provides aread-out of receptor activation. (Binkowski, B. F. et al. “A LuminescentBiosensor with Increased Dynamic Range for Intracellular cAMP.” AcsChemical Biology 6, 1193-1197 (2011).) Consistent with previous reports,PTH(1-34)-NH₂ is very active in both assays (EC₅₀=0.38 nM for PTHR1, andEC₅₀=0.87 nM for PTHR2). (Carter, P. H. et al. Actions of the SmallMolecule Ligands SW106 and AH-3960 on the Type-1 Parathyroid HormoneReceptor. Molecular Endocrinology 29, 307-321 (2015).)

For PTHR1 activation, different patterns of substitution tolerance wereobserved among the α→β² and α→β³ replacements. All three isomeric βresidues were well-tolerated in place of Ser-1 or Leu-7, and none of thethree was tolerated in place of Ser-3 or Glu-4. At the remainingpositions, variable responses to α→β replacement were observed. Thus,for Val-2, the β³ replacement has little effect on agonist potency, butboth β² replacements cause significant declines in potency. For Ile-5,the β³ and (R*)-β² replacements cause modest activity declines, but the(S*)-β² replacement matches PTH(1-34)-NH₂ in activity. Use of(S*)-β²-hGln at position 6 has no effect on agonist activity, butplacing either β³-hGln or (R*)-β²-hGln at this site causes a substantialactivity decline. Both enantiomers of β²-hNle are well-tolerated inplace of Met-8, but use of either β³-hMet or β³-hNle at this positioncauses a substantial decline in activity. The overall trend among α→β³replacements is consistent with a previously reported β³ scan of PTH(1-34)-NH₂.²⁸

The PTHR2 assay displayed a greater sensitivity to α→β replacements thandid the PTHR1 assay. For several single-β substitutions, the decline inagonist activity was so profound that an EC₅₀ value could not bedetermined. In contrast to the findings with PTHR1, there was noposition among the first eight residues of PTH(1-34)-NH₂ at which allthree isomeric α→β replacements were well tolerated. At position 1, the(S*)-β² and β³ replacements had little effect on agonist potency, butthe (R*)-β² replacement caused a modest decline. At position 2, the(S*)-β² replacement caused a modest activity decline, while the (R*)-β²and β³ replacements were well tolerated. At the remaining sites, the(R*)-β² replacements were uniformly unfavorable in terms of agonistpotency, while the impact of (S*)-β² replacements was quite variable,ranging from very disruptive (position 4) to well tolerated (positions 6and 7). β³ replacements at positions 3-8 of PTH(1-34)-NH₂ exertedvariable effects on PTHR2 agonist activity as well, but the patterndiffered from that manifested among the (S*)-β² replacements.

Enhanced Selectivity Via Double α→β Replacement.

Based on the β scan results summarized in Tables 2 and 3, it appearedpossible to design PTH(1-34)-NH₂ homologues containing α→β replacementsat two sites in the N-terminal region that would display highselectivity for either PTHR1 activation or PTHR2 activation, in contrastto the potent activation of both receptors displayed by PTH(1-34)-NH₂itself. As a PTHR1-selective candidate, we examined α/β-peptide 14 (SEQ.ID. NO: 14) which contains α→(R*)-β² replacements at positions 1 and 7.α/β-Peptide 15 (SEQ. ID. NO: 15) containing α→(R*)-β² replacement atposition 2 and α→β³ replacement at position 6, was evaluated as aPTHR2-selective candidate. The basis for these replacement choices isshown in Table 4.

Green denotes potent PTHR1-selective agonism; yellow denotes potentPTHR2-selective agonism; purple denotes potent agonism of both PTHR1 andPTHR2. Data represent mean±s.e.m for >3 independent measurements. #β²-hNle was used instead of β²-hMet; both β³-hMet and β³-hNle wereevaluated at the 8^(th) position; the value shown is the EC₅₀ ofβ³-hMet.

Results summarized in Table 5 show that for both 14 and 15, the two α→βreplacements function synergistically.

TABLE 5 Signaling and binding properties of PTHR1- and PTHR2-selectiveα/β- peptide analogues of PTH(1-34)-NH₂. EC₅₀, E_(max), R⁰/RG IC₅₀values for PTH(1-34)- NH₂, α/β-peptide 14, and α/β-peptide 15. PTHR1PTHR2 Peptide EC₅₀ (nM) E_(max) R⁰-IC₅₀ (nM) RG-IC₅₀ (nM) EC₅₀ (nM)E_(max) R⁰-IC₅₀ (nM) RG-IC₅₀ (nM) PTH(1-34)-NH₂ 0.22 ± 0.04 100 6.95 ±0.81 0.34 ± 0.06 1.07 ± 0.14 100 0.96 ± 0.48 0.63 ± 0.23 a/b-peptide 140.23 ± 0.04 100 ± 1 27.18 ± 7.22  0.13 ± 0.02 ND ND 4.94 ± 0.81 0.04 ±0.01 a/b-peptide 15 22.09 ± 1.50   79 ± 6 5.25 ± 1.64 0.80 ± 0.08 0.13 ±0.01 104 ± 2 0.64 ± 0.03 0.84 ± 0.19

FIGS. 10A and 10B depict dose-response curves of PTHR1 activation (FIG.10A) and PTHR2 activation (FIG. 10B) in HEK293 cells stably expressingPTHR1 or PTHR2. For PTHR1 activation and all the binding data, datarepresent mean±s.e.m from three independent measurements; For PTHR2activation, data represent mean±s.e.m from four independentmeasurements. Curves were fit to the data using a four-parametersigmoidal dose-response equation.

On one hand, neither replacement in 14, on its own, causes a diminutionof PTHR1 agonist activity (Table 2), and implementing the tworeplacements simultaneously also does not cause an activity decline. Onthe other hand, each of the replacements in 14 leads to significantdecline in PTHR2 agonist activity (˜6-fold and ˜40-fold), and thepairing generates an α/β-peptide that has almost no detectable activityfor this receptor in this assay. For 15, the α→β replacementsindividually cause slight increases in agonist activity at PTHR2, andthe combination of these backbone modifications leads to further potencyenhancement relative to PTH(1-34)-NH₂. But, each of the replacements inα/β-peptide 15 causes a significant decline in PTHR1 agonist potency(˜10-fold and ˜20-fold), and the combination leads to a more substantialpotency decline (˜100-fold). Moreover, 15 reaches a maximum PTHR1activation level that is only ˜80% of the maximum achieved byPTH(1-34)-NH₂.

Binding to PTHR1 and PTHR2.

The selective agonism displayed by α/β-peptides 14 and 15 relative toPTH(1-34)-NH₂ (SEQ. ID. NO: 16) could arise because of differencesrelative to PTH(1-34)-NH₂ in their affinities for PTHR1 and PTHR2, orbecause of differences in the abilities of 14 and 15 to activate eachreceptor upon binding. We conducted binding assays for PTHR1 and PTHR2with these two PTH(1-34)-NH₂ analogues in an effort to distinguish thesetwo possibilities. See Table 5. Two conformational states have beenproposed for PTHR1 and for PTHR2, one that is G protein-dependent (RG)and another that is G protein-independent (R⁰).⁵ Distinct assays areavailable for binding to the R⁰ and RG states for each receptor. Forboth PTHR1 and PTHR2, α/β-peptide 14 has higher RG affinity and lower R⁰affinity than does PTH (1-34)-NH₂. In contrast, α/β-peptide 15 has lowerRG affinity than does PTH(1-34)-NH₂ for both receptors, but 15 andPTH(1-34)-NH₂ are comparable in terms of R⁰ affinity for both receptors.The PTHR1/PTHR2 R⁰ affinity ratios are very similar for 14, 15 andPTH(1-34)-NH₂, and the PTHR1/PTHR2 RG affinity ratios vary only byapproximately six-fold among these three agonists.

Collectively, these observations indicate that the selective agonismdisplayed by each of the α/β peptides arises mainly from selectivereceptor activation rather than from selective binding to one receptoror the other. Further evidence that a backbone-modified PTH(1-34)-NH₂homologue can maintain affinity for a receptor despite loss of agonistactivity was obtained from the observation that α/β-peptide 14 functionsas an antagonist of PTHR2 activation by PTH(1-34)-NH₂. See FIG. 11. Theconclusion that backbone-modified homologues of PTH(1-34)-NH₂ retain theability to occupy the orthosteric site but are deficient in terms ofinducing an active receptor conformation stands in contrast to previousanalysis of naturally selective agonists PTHrP and TIP39, for whichselectivity in receptor binding plays a major role in determiningagonist selectivity. See Hoare, S. R. J., Clark, J. A. & Usdin, T. B.Molecular determinants of tuberoinfundibular peptide of 39 residues(TIP39) selectivity for the parathyroid hormone-2 (PTH2)receptor—N-terminal truncation of TIP39 reverses PTH2 receptor/PTH1receptor binding selectivity. Journal of Biological Chemistry 275,27274-27283 (2000) and Gardella, T. J., Luck, M. D., Jensen, G. S.,Usdin, T. B. & Juppner, H. Converting parathyroid hormone-relatedpeptide (PTHrP) into a potent PTH-2 receptor agonist. Journal ofBiological Chemistry 271, 19888-19893 (1996).

Molecular Basis of PTHR1 vs. PTHR2 Selectivity.

Previous receptor-mutation studies have identified sites within PTHR1and PTHR2 that play critical roles in determining peptide agonistselectivity. (Bergwitz, C., Jusseaume, S. A., Luck, M. D., Juppner, H. &Gardella, T. J. Residues in the membrane-spanning and extracellular loopregions of the parathyroid hormone (PTH)-2 receptor determine signalingselectivity for PTH and PTH-related peptide. Journal of BiologicalChemistry 272, 28861-28868 (1997).) For example, PTHrP (1-36)-NH₂ is apotent agonist of PTHR1 and a very weak agonist of PTHR2; however,modifications at three sites in PTHR2, based on residues found atanalogous sites in PTHR1, rescue the agonist activity of PTHrP(1-36)-NH₂. Specifically, any of three modifications to the PTHR2sequence, (i) replacement of residues 199-20 at the N-terminal side ofextracellular loop 1 (ECL1) of PTHR2 with the corresponding segment ofPTHR1, (ii) mutation of 1244 to L, or (iii) mutation of Y318 to I,generates a PTHR2 variant that is much more susceptible to activation byPTHrP (1-36)-NH₂ relative to wide-type PTHR2 (data not shown).Evaluation of α/β-peptide 14 with this panel of three PTHR2 variantsyields an activity profile that is distinct from the activity profileobserved with PTHrP(1-36)-NH₂. At the level of receptor expressionrequired to detect significant activity rescue for 100 nMPTHrP(1-36)-NH₂ at the three mutant receptors, substantial activity atwild-type PTHR2 is observed for 100 nM α/β-peptide 14. Significantincreases in agonist activity are observed for two of the PTHR2 variantsrelative to wild-type receptor (ECL1 chimera and Y318I), but a decreaseis evident relative to wild-type receptor for the third PTHR2 variant,I244L. Detailed interpretation of these differences is not possible inthe absence of atomic-resolution structural data for PTHR1 or PTHR2, butthe distinct response profiles of PTHrP(1-36)-NH₂ and α/β-peptide 14 tothis set of receptor variants, particularly I244L, suggest that themolecular determinants underlying the PTHR1 vs. PTHR2 selectivity are atleast partially different between PTHrP(1-36)-NH₂ and α/β-peptide 14.

Reciprocal point mutations of PTHR1 were investigated to see if theywould enhance the signaling activity of PTHR2-selective agonists.TIP39-NH₂ does not activate wild-type PTHR1 and appears to be a veryweak agonist of mutants PTHR1-L2891 and PTHR1-I363Y (data not shown).α/β-Peptide 15 deviates partially from this pattern in that PTHR1-I363Yis even less susceptible to activation by 15 than is wild-type PTHR1.Overall, the observations with receptor variants suggest that theresponse of PTHR1 and PTHR2 to agonists that are selective by virtue ofside chain identity (such as PTHrP (1-36)-NH₂ or TIP39-NH₂) involves aset of contact residues on the receptor that is partially distinct fromthose that mediate the response to agonists that are selective by virtueof backbone modification (such as α/β-peptides 14 and 15).

Duration of Activation for PTHR1 and PTHR2.

To assess the duration of PTHR1 and PTHR2 activation induced byα/β-peptides 14 and 15, the time course of cAMP production by eachreceptor was examined after stimulation with an agonist and subsequentwashing of the cells to remove unbound peptide (wash-out assay).PTH(1-34)-NH₂ and the naturally selective agonists PTHrP(1-36)-NH₂ andTIP39-NH₂ were used as controls. During the initial “ligand-on” phase,cells stably expressing PTHR1 or PTHR2 were stimulated with an agonistconcentration corresponding to ˜EC₈₀; the medium contained D-luciferin.The luminescence emission caused by cAMP production was monitored untileach agonist reached maximum response (E_(max); ˜14 min in each case),at which point unbound peptide was washed away. After the addition offresh medium containing D-luciferin, the luminescence decay is measured.The area under the “ligand-off” curve (AUC) reflects the duration ofsignaling, which may be related to residence time of the agonist on thereceptor but could have other origins at the molecular level. At PTHR1,the selective agonists α/β-peptide 14 and PTHrP(1-36)-NH₂ lead to moretransient signaling (smaller AUC) than does PTH(1-34)-NH₂. See TableS1a.6 At PTHR2, the selective agonists α/β-peptide 15 and TIP39-NH₂induce more prolonged signaling than does PTH(1-34)-NH₂. See Table S1b.7

Data represent mean±s.e.m from 10 independent experiments. b. PTHR2signaling durations of PTH(1-34)-NH₂, TIP39-NH₂, and α/β-peptide 2. Datarepresent mean±s.e.m from 11 independent experiments

TABLE 6 PTHR1 signaling durations of PTH(1-34)-NH₂, PTHrP(1-36)-NH₂, andα/β-peptide 14. Normalized ligand-on Normalized ligand-off PeptideE_(max) AUC PTH(1-34)-NH₂ 100 100 PTHrP(1-36)-NH₂ 102 ± 1 55 ± 2α/β-peptide 14  99 ± 2 68 ± 3 Data represent mean ± s.e.m from 10independent experiments.

TABLE 7 PTHR2 signaling durations of PTH(1-34)-NH₂, TIP39-NH₂, and α/β-peptide 2. Normalized ligand-on Normalized ligand-off Peptide E_(max)AUC PTH(1-34)-NH₂ 100 100 TIP39-NH₂  99 ± 1 226 ± 22 α/β-peptide 1 103 ±1 197 ± 16 Data represent mean ± s.e.m from 11 independent experiments.

EC50 data for the complete series of systematic substitutions is shownin Table 8

TABLE 8  Agonist activity toward hPTHR1 and hPTHR2 PTHR1 EC50 PTHR1 EC50Polypeptide (nM) (nM) PTH:  0.38 ± 0.04  0.87 ± 0.09 (SEQ. ID. NO: 16)SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF-NH₂ (SEQ. ID. NO: 22)SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF-NH₂  0.35 ± 0.06  5.97 ± 1.21(SEQ. ID. NO: 23) SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF-NH₂  4.47 ± 0.47 0.21 ± 0.03 (SEQ. ID. NO: 24) SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF-NH₂29.96 ± 6.33 >100  (SEQ. ID. NO: 25)SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF-NH₂ 22.90 ± 1.89 >1000(SEQ. ID. NO: 26) SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF-NH₂  2.24 ±0.21 >100  (SEQ. ID. NO: 27) SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF-NH₂ 7.83 ± 0.40 51.94 ± 8.82 (SEQ. ID. NO: 28)SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF-NH₂  0.36 ± 0.11 41.45 ± 5.18(SEQ. ID. NO: 29) SVSEIQL 

 HNLGKHLNSMERVEWLRKKLQDVHNF-NH₂  0.40 ± 0.07 >1000 (SEQ. ID. NO: 30) SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF-NH₂  1.55 ± 0.30  1.55 ± 0.30(SEQ. ID. NO: 31) S V SEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF-NH₂  4.72 ± 1.04 4.72 ± 1.04 (SEQ. ID. NO: 32) SV S EIQLMHNLGKHLNSMERVEWLRKKLQDVHNF-NH₂ 8.36 ± 0.69  8.36 ± 0.69 (SEQ. ID. NO: 33) SVS EIQLMHNLGKHLNSMERVEWLRKKLQDVHNF-NH₂ >100 >100  (SEQ. ID. NO: 34) SVSE IQLMHNLGKHLNSMERVEWLRKKLQDVHNF-NH₂ 14.99 ± 3.70 14.99 ± 3.70(SEQ. ID. NO: 35) SVSEI Q LMHNLGKHLNSMERVEWLRKKLQDVHNF-NH₂  1.96 ± 0.45 1.96 ± 0.45 (SEQ. ID. NO: 36) SVSEIQ L MHNLGKHLNSMERVEWLRKKLQDVHNF-NH₂ 0.24 ± 0.01  0.24 ± 0.01 (SEQ. ID. NO: 37) SVSEIQL 

 HNLGKHLNSMERVEWLRKKLQDVHNF-NH₂ 10.31 ± 1.65 10.31 ± 1.65(SEQ. ID. NO: 38) S VSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF-NH₂  0.27 ± 0.06 0.55 ± 0.16 (SEQ. ID. NO: 39) S V SEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF-NH₂ 0.55 ± 0.09  0.80 ± 0.14 (SEQ. ID. NO: 40) SV SEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF-NH₂  8.29 ± 0.09  2.37 ± 0.15(SEQ. ID. NO: 41) SVS E IQLMHNLGKHLNSMERVEWLRKKLQDVHNF-NH₂ 10.36 ± 1.8760.11 ± 9.82 (SEQ. ID. NO: 42) SVSE I QLMHNLGKHLNSMERVEWLRKKLQDVHNF-NH₂ 3.40 ± 1.22  9.50 ± 1.67 (SEQ. ID. NO: 43) SVSEI QLMHNLGKHLNSMERVEWLRKKLQDVHNF-NH₂  8.45 ± 1.35  0.18 ± 0.02(SEQ. ID. NO: 44) SVSEIQ L MHNLGKHLNSMERVEWLRKKLQDVHNF-NH₂  0.84 ± 0.1235.04 ± 2.84 (SEQ. ID. NO: 45) SVSEIQL M HNLGKHLNSMERVEWLRKKLQDVHNF-NH₂20.99 ± 2.30 >100 

Nutritional Compositions:

The present disclosure includes nutritional compositions. Suchcompositions include any food or preparation for human consumption(including for enteral or parenteral consumption) which when taken intothe body (a) serve to nourish or build up tissues or supply energyand/or (b) maintain, restore or support adequate nutritional status ormetabolic function.

The nutritional composition comprises at least one PTH analog asdescribed herein and may either be in a solid or liquid form.Additionally, the composition may include edible macronutrients,vitamins and minerals in amounts desired for a particular use. Theamount of such ingredients will vary depending on whether thecomposition is intended for use with normal, healthy infants, childrenor adults having specialized needs such as those which accompanyhyperglycemic metabolic conditions.

Examples of macronutrients which may be added to the composition includebut are not limited to edible fats, carbohydrates and proteins. Examplesof such edible fats include but are not limited to coconut oil, soy oil,and mono- and diglycerides. Examples of such carbohydrates include butare not limited to glucose, edible lactose and hydrolyzed starch.Additionally, examples of proteins which may be utilized in thenutritional composition include but are not limited to soy proteins,electrodialysed whey, electrodialysed skim milk, milk whey, or thehydrolysates of these proteins.

With respect to vitamins and minerals, the following may be added to thenutritional compositions described herein: calcium, phosphorus,potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc,selenium, iodine, and Vitamins A, E, D, C, and the B complex. Other suchvitamins and minerals may also be added.

Examples of nutritional compositions disclosed herein include but arenot limited to infant formulas, dietary supplements, dietarysubstitutes, and rehydration compositions. Nutritional compositions ofparticular interest include but are not limited to those utilized forenteral and parenteral supplementation for infants, specialist infantformulas, supplements for the elderly, and supplements for those withhyperglycemia.

The nutritional composition of the present invention may also be addedto food even when supplementation of the diet is not required. Forexample, the composition may be added to food of any type including butnot limited to margarines, modified butters, cheeses, milk, yoghurt,chocolate, candy, snacks, salad oils, cooking oils, cooking fats, meats,fish and beverages.

In a preferred version, the nutritional composition is an enteralnutritional product, more preferably, an adult or pediatric enteralnutritional product. This composition may be administered to adults orchildren experiencing stress or having specialized needs due to chronicor acute disease states. The composition may comprise, in addition toPTH(1-34) analogs described herein, macronutrients, vitamins andminerals as described above. The macronutrients may be present inamounts equivalent to those present in human milk or on an energy basis,i.e., on a per calorie basis.

Methods for formulating liquid or solid enteral and parenteralnutritional formulas are well known in the art. An enteral formula, forexample, may be sterilized and subsequently utilized on a ready-to-feed(RTF) basis or stored in a concentrated liquid or powder. The powder canbe prepared by spray drying the formula prepared as indicated above, andreconstituting it by rehydrating the concentrate. Adult and pediatricnutritional formulas are well known in the art and are commerciallyavailable (e.g., Similac®-brand and Ensure®-brand formulas from RossProducts Division, Abbott Laboratories, Columbus, Ohio). A PTH(1-34)analog produced in accordance with the present disclosure may be addedto commercial formulas of this type.

The energy density of the nutritional compositions in liquid form mayrange from about 0.6 Kcal to about 3 Kcal per ml. When in solid orpowdered form, the nutritional supplements may contain from about 1.2 tomore than 9 Kcals per gram, preferably about 3 to 7 Kcals per gm. Ingeneral, the osmolality of a liquid product should be less than 700 mOsmand, more preferably, less than 660 mOsm.

Pharmaceutical Compositions:

Also disclosed herein are pharmaceutical compositions comprising one ormore of the PTH analogs or a pharmaceutically suitable salt thereof asdescribed herein. More specifically, the pharmaceutical composition maycomprise one or more of the PTH analogs as well as a standard,well-known, non-toxic pharmaceutically suitable carrier, adjuvant orvehicle such as, for example, phosphate buffered saline, water, ethanol,polyols, vegetable oils, a wetting agent or an emulsion such as awater/oil emulsion. The composition may be in either a liquid, solid orsemi-solid form. For example, the composition may be in the form of atablet, capsule, ingestible liquid or powder, injectible, suppository,or topical ointment or cream. Proper fluidity can be maintained, forexample, by maintaining appropriate particle size in the case ofdispersions and by the use of surfactants. It may also be desirable toinclude isotonic agents, for example, sugars, sodium chloride, and thelike. Besides such inert diluents, the composition may also includeadjuvants, such as wetting agents, emulsifying and suspending agents,sweetening agents, flavoring agents, perfuming agents, and the like.

Suspensions, in addition to the active compounds, may comprisesuspending agents such as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanthor mixtures of these substances.

Solid dosage forms such as tablets and capsules can be prepared usingtechniques well known in the art of pharmacy. For example, PTH analogsproduced as described herein can be tableted with conventional tabletbases such as lactose, sucrose, and cornstarch in combination withbinders such as acacia, cornstarch or gelatin, disintegrating agentssuch as potato starch or alginic acid, and a lubricant such as stearicacid or magnesium stearate. Capsules can be prepared by incorporatingthese excipients into a gelatin capsule along with antioxidants and therelevant PTH analog.

For intravenous administration, the analogs may be incorporated intocommercial formulations such as Intralipid©-brand fat emulsions forintravenous injection. (“Intralipid” is a registered trademark ofFresenius Kabi AB, Uppsalla, Sweden.) Where desired, the individualcomponents of the formulations may be provided individually, in kitform, for single or multiple use. A typical intravenous dosage of arepresentative PTH analog as described herein is from about 0.1 mg to100 mg daily and is preferably from 0.5 mg to 20 mg daily. Dosages aboveand below these stated ranges are specifically within the scope of theclaims.

Possible routes of administration of the pharmaceutical compositionsinclude, for example, enteral (e.g., oral and rectal) and parenteral.For example, a liquid preparation may be administered, for example,orally or rectally. Additionally, a homogenous mixture can be completelydispersed in water, admixed under sterile conditions withphysiologically acceptable diluents, preservatives, buffers orpropellants in order to form a spray or inhalant. The route ofadministration will, of course, depend upon the desired effect and themedical stated of the subject being treated. The dosage of thecomposition to be administered to the patient may be determined by oneof ordinary skill in the art and depends upon various factors such asweight of the patient, age of the patient, immune status of the patient,etc., and is ultimately at the discretion of the medical professionaladministering the treatment.

With respect to form, the composition may be, for example, a solution, adispersion, a suspension, an emulsion or a sterile powder which is thenreconstituted. The composition may be administered in a single dailydose or multiple doses.

The present disclosure also includes treating hypoparathyroid disordersin mammals, including humans, by administering ananti-hypoparathyroid-effective and/or PTHR agonist-effective amount ofone or more the PTH analogs described herein. In particular, thecompositions of the present invention may be used to treathypoparathyroid conditions of any and all description.

It should be noted that the above-described pharmaceutical andnutritional compositions may be utilized in connection with non-humananimals, both domestic and non-domestic, as well as humans.

Peptide Synthesis and Purification:

Peptides were synthesized as C-terminal amides on NovaPEG rink amideresin (EMD-Millipore, Billerica, Mass.) using previously reportedmicrowave-assisted solid-phase conditions based on Fmoc protection ofmain chain amino groups.³¹ Briefly, protected amino acids were activatedwith 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HBTU) and N-hydroxybenzotriazole (HOBt) in thepresence of N,N-diisopropylethylamine (DIEA). The growing peptide chainwas deprotected using 20% piperdine in DMF. Protected β³-homoamino acidswere purchased from PepTech Corporation (Bedford, Mass.).

After synthesis, the peptides were cleaved from the resin and sidechains were deprotected using reagent K (82.5% TFA, 5% phenol, 5% H₂O,5% thioanisole, 2.5% ethanedithiol)³¹ for two hours. The TFA solutionwas dripped into cold diethyl ether to precipitate the deprotectedpeptide. Peptides were purified on a prep-C18 column using reversephase-HPLC. Purity was assessed by analytical RP-HPLC (solvent A: 0.1%TFA in water, solvent B: 0.1% TFA in acetonitrile, C18 analytical column(4.6×250 mm), flow rate 1 mL/min, gradient 10-60% B solvent over 50minutes). Masses were measured by MALDI-TOF-MS.

Protease Stability:

An HPLC assay was used to assess proteolytic stability.^(30,32) Peptideconcentration was determined by UV-Vis spectroscopy (calculated from theUV-vis absorption at 280 nm, ε_(280 nm)=5,690 M⁻¹cm⁻¹ for all peptidesexcept D6 and D7, ε_(280 nm)=11,380 M⁻¹cm⁻¹ based on an extinctioncoefficient for the tryptophan sidechain chromophore of 5,690 M⁻¹cm⁻¹).³³ Peptide stock solutions were prepared in degassed water to aconcentration of 200 μM. Sequencing-grade trypsin from bovine pancreaswas purchased from Sigma Aldrich (St. Louis, Mo.) and prepared to astock concentration of 100 μg/mL in 1 mM HCl. The protease reaction wascarried out in 0.6 mL Eppendorf tubes at room temperature. The reactionsolution was prepared by combining 40 μL of 200 μM peptide (finalconcentration 40 μM), 20 μL of 10×TBS pH 8.5 (final concentration 15 mMTris, 150 mM NaCl, or 1×TBS), 130 μL of water, and 10 μL of 100 μg/mLprotease (added last, final concentration 5 μg/mL, in a total volume of200 μL). Each proteolysis experiment was run in duplicate. Followingaddition of protease, the reaction was timed and quenched by combining a50 μL aliquot of the proteolysis mixture with 50 μL of 0.1%trifluoroacetic acid in acetonitrile. A portion (75 μL) of the quenchedreaction mixture was injected onto an analytical RP-HPLC (see peptidesynthesis and purification section above), and peaks were analyzed. Thetime course of peptide degradation was experimentally determined byintegrating the area of the peak corresponding to the non-hydrolyzedpeptide in a series of HPLC traces, with duplicate proteolysis reactionsbeing used to generate error bars corresponding to the standarddeviation. The final 50 μL of the reaction solution was used to acquireMALDI-TOF mass spectrometry data for identification of peptide fragmentsresulting from proteolysis. Proteolysis was observed at all predictedtrypsin cut sites for PTH(1-34). Shown in FIGS. 5A, 5B, and 5C are timecourse data for peptide degradation. Exponential decay curves andhalf-life values were generated using GraphPad Prism version 5.0(GraphPad Software, La Jolla, Calif.).

Binding and cAMP Dose Response:

Reported IC₅₀ and EC₅₀ values are the average of ≥4 independentmeasurements. Each assay comprises ≥7 data points (differentconcentrations) per α/β-peptide, with each data point representing theaverage from duplicate wells. Binding to the RG and R⁰ conformations ofthe human or rat PTHR-1 was assessed by competition assays performed in96-well plates by using membranes from transiently transfected COS-7cells as previously described.^(6,7) In brief, binding to R⁰ wasassessed by using ¹²⁵I-PTH(1-34) as tracer radio-ligand and includingGTPγS in the reaction (1×10⁻⁵ M). Binding to RG was assessed by usingmembranes containing a high-affinity, negative-dominant G_(α)S subunit(G_(α)S ND)¹⁵, and ¹²⁵I-M-PTH(1-15)⁶ as tracer radio-ligand.

cAMP signaling was assessed using HEK-293-derived cell lines that stablyexpress the GloSensor™-brand cAMP reporter (Promega Corp.) along witheither the WT human PTHR-1 (GP-2.3 cells) or WT rat PTHR-1 (GR-35cells). For cAMP dose-response assays, monolayers of confluent HEK 293cells were pre-incubated with buffer containing d-luciferin (0.5 mM) in96 well plates at room temperature until a stable baseline level ofluminescence was established (30 min). Varying concentrations of agonistwere then added, and the time course of luminescence response wasrecorded using a Perkin Elmer plate reader following α/β-peptideaddition. The maximal luminescence response (observed 12-16 min afterligand addition) was used for generating dose response curves.

“Washout” Assays:

The duration of PTH analog stimulated cAMP response following removal ofthe solution containing dissolved peptide from the confluent HEK293 cellmonolayers has been shown to be predictive of the duration of in vivocalcemic responses in mice.⁸ This parameter of in vitro PTH analogperformance shows strong positive correlation with R⁰ binding affinity(high R⁰ binding affinity correlates with prolonged cAMP signalingfollowing washout). Washout assays were carried out for PTH(1-34),LA-PTH, and Ligands 8, 10, 12, and 100 to assess the contribution ofaltered R⁰ binding affinity to the duration of calcemic responsesobserved in vivo. M-PTH(1-34) (data not shown), a sidechain-altered PTHanalog that has previously been shown to induce prolonged responsesfollowing washout and prolonged calcemic response in vivo withnative-like bioavailability, was included as a control. HEK293 cellmonolayers expressing rat PTHR-1 (GR35 cells) were treated with ligandin for 14 minutes. This buffer was then discarded, and the cellmonolayer was rinsed (2×). Buffer containing luciferin was introducedfor 120 minutes. Luminescence response was recorded before and afterwashout, with luminescence readings recorded every 2 minutes. The areaunder the curve (AUC) for luminescence response curve was determinedusing GraphPad Prism.

In Vivo Pharmacology: Calcemic Response:

PTH(1-34) causes a transient rise in the blood concentration of Ca²⁺,which peaks after about one hour.⁸ Okazaki et al. have proposed that thecalcemic effect duration resulting from injection of a PTHR-1 agonist iscontrolled, at least in part, by affinity for the R⁰ state of thereceptor.⁸ According to this hypothesis, agonists with high R⁰ affinitycan remain bound to the receptor through multiple cycles of G_(α)binding and release, which should induce prolonged signaling.⁷ Thepathways by which PTH is removed from circulation have not been fullyelucidated, but enzymatic degradation probably contributes to the rapiddisappearance of PTH(1-34) in vivo.²⁰

To evaluate calcemic response, mice (C57BL/6, male, age 9-12 weeks) aretreated in accordance with the ethical guidelines adopted byMassachusetts General Hospital. Mice (n=5 per compound) and are injectedsubcutaneously with vehicle (10 mM citric acid/150 mM NaCl/0.05%Tween-80, pH 5.0) or vehicle containing PTH(1-34) or one of the ligandsdisclosed herein at a dose of 20 nmol/kg body weight. Blood is withdrawnjust prior to injection (t=0) or at times thereafter. Tail vein blood iscollected and immediately used for analysis. Blood Ca²⁺ concentration ismeasured with a Chiron Diagnostics model 634 Ca²⁺/pH analyzer.

In Vivo Pharmacokinetics:

Blood content of the ligands described herein analog is assessed inplasma from mice injected with vehicle or vehicle containing a ligand asdescribed herein at a dose of 20 nmol/kg body weight in an experimentperformed separately from the calcemic response assays described above.Blood is withdrawn just prior to injection (t=0) or at times thereafter.Tail vein blood is collected and treated with protease inhibitors(aprotinin, leupeptin, EDTA), centrifuged to remove blood cells, mixedwith cAMP response assay buffer, and administered to GP2.3 cells. Theraw luminescence readouts recorded in this assay are converted to bloodpeptide concentrations through use of a standard curve relatingluminescence response to known peptide concentrations under identicalassay conditions.

Data Calculations:

Data were processed by using the Microsoft Excel and GraphPad Prism 4.0software packages. Data from binding and cAMP dose-response assays wereanalyzed using a sigmoidal dose-response model with variable slope.Washout responses were assessed by quantifying the area under theluminescence response curve (AUC) following washout. Post-washout AUCwas normalized by dividing the post-washout AUC by the pre-washout AUC.Paired data sets were statistically compared by using Student's t test(two-tailed) assuming unequal variances for the two sets.

REFERENCES CITED

The following documents are incorporated herein by reference.

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What is claimed is:
 1. An isolated, unnatural peptide analoguecomprising: PTH, a parathyroid hormone receptor (PTHR-1, PTHR-2)agonist-effective fragment of PTH, a parathyroid hormone related protein(PTHrP), a PTHR-1 or PTHR-2 agonist-effective fragment of PTHrP, M-PTH,a PTHR-1 or PTHR-2 agonist-effective fragment of M-PTH, BA058, or aPTHR-1 or PTHR-2 agonist-effective fragment of BA058, in which at leastone naturally occurring (L)-α-amino acid residue at position 6, 7, or 8from the N-terminus is replaced with a β-amino acid residue or a(D)-α-amino acid; and salts thereof.
 2. The peptide analogue of claim 1,wherein the at least one naturally occurring (L)-α-amino acid residue atposition 6, 7, or 8 from the N-terminus is replaced with a β²-amino acidresidue.
 3. The peptide analogue of claim 2, wherein the at least onenaturally occurring (L)-α-amino acid residue at 6, 7, or 8 from theN-terminus is replaced with a β²-amino acid residue having a side-chainidentical to the (L)-α-amino acid residue it replaces.
 4. The peptideanalogue of claim 1, wherein the at least one naturally occurring(L)-α-amino acid residue at 6, 7, or 8 from the N-terminus is replacedwith a β³-amino acid residue.
 5. The peptide analogue of claim 4,wherein the at least one naturally occurring (L)-α-amino acid residue at6, 7, or 8 from the N-terminus is replaced with a β³-amino acid residuehaving a side-chain identical to the (L)-α-amino acid residue itreplaces.
 6. The peptide analogue of claim 1, wherein the at least onenaturally occurring (L)-α-amino acid residue at 6, 7, or 8 from theN-terminus is replaced with a (D)-α-amino acid.
 7. The peptide analogueof claim 6, wherein the at least one naturally occurring (L)-α-aminoacid residue at 6, 7, or 8 from the N-terminus is replaced with a(D)-α-amino acid residue having a side-chain identical to the(L)-α-amino acid residue it replaces.
 8. The peptide analogue of claim1, selected from the group consisting of (SEQ. ID. NO: 8)SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF (SEQ. ID. NO: 10) SVSEIQ

MHNLGKHLNSMERVEWLRKKLQDVHNF and (SEQ. ID. NO: 12) SVSEIQL^(n)LHNLGKHLNSMERVEWLRKKLQDVHNF (SEQ. ID. NO: 14)SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF (SEQ. ID. NO: 15) SVSEI QLMHNLGKHLNSMERVEWLRKKLQDVHNF

wherein L=(R*) β² leucine; L=(D)-leucine; and ^(n)L=(R*)-β²-norleucine.Q=β³ glutamine S=β² serine.
 9. The peptide analogue of claim 1, in whichat least one additional naturally occurring (L)-α-amino acid residue atposition 1 or 2 from the N-terminus is replaced with a β-amino acidresidue or a (D)-α-amino acid.
 10. An isolated, unnatural peptideanalogue consisting of: PTH, a parathyroid hormone receptor (PTHR-1,PTHR-2) agonist-effective fragment of PTH, a parathyroid hormone relatedprotein (PTHrP), a PTHR-1 or PTHR-2 agonist-effective fragment of PTHrP,M-PTH, a PTHR-1 or PTHR-2 agonist-effective fragment of M-PTH, BA058, ora PTHR-1 or PTHR-2 agonist-effective fragment of BA058, in which atleast one naturally occurring (L)-α-amino acid residue at position 6, 7,or 8 from the N-terminus is replaced with a β-amino acid residue or a(D)-α-amino acid; and salts thereof.
 11. The peptide analogue of claim10, wherein the at least one naturally occurring (L)-α-amino acidresidue at position 6, 7, or 8 from the N-terminus is replaced with aβ²-amino acid residue.
 12. The peptide analogue of claim 11, wherein theat least one naturally occurring (L)-α-amino acid residue at position 6,7, or 8 from the N-terminus is replaced with a β²-amino acid residuehaving a side-chain identical to the (L)-α-amino acid residue itreplaces.
 13. The peptide analogue of claim 10, wherein the at least onenaturally occurring (L)-α-amino acid residue at position 6, 7, or 8 fromthe N-terminus is replaced with a β³-amino acid residue.
 14. The peptideanalogue of claim 13, wherein the at least one naturally occurring(L)-α-amino acid residue at position 6, 7, or 8 from the N-terminus isreplaced with a β³-amino acid residue having a side-chain identical tothe (L)-α-amino acid residue it replaces.
 15. The peptide analogue ofclaim 10, wherein the at least one naturally occurring (L)-α-amino acidresidue at position 6, 7, or 8 from the N-terminus is replaced with a(D)-α-amino acid.
 16. The peptide analogue of claim 15, wherein the atleast one naturally occurring (L)-α-amino acid residue at position 6, 7,or 8 from the N-terminus is replaced with a (D)-α-amino acid residuehaving a side-chain identical to the (L)-α-amino acid residue itreplaces.
 17. The peptide analogue of claim 10, selected from the groupconsisting of (SEQ. ID. NO: 8) SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF (SEQ.ID. NO: 10) SVSEIQ

MHNLGKHLNSMERVEWLRKKLQDVHNF and (SEQ. ID. NO: 12) SVSEIQL^(n)LHNLGKHLNSMERVEWLRKKLQDVHNF (SEQ. ID. NO: 14)SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF (SEQ. ID. NO: 15) SVSEI QLMHNLGKHLNSMERVEWLRKKLQDVHNF

wherein L=(R*) β² leucine; L=(D)-leucine; and ^(n)L=(R*)-β²-norleucine.Q=β³ glutamine S=β² serine.
 18. The peptide analogue of claim 10, inwhich at least one additional naturally occurring (L)-α-amino acidresidue at position 1 or 2 from the N-terminus is replaced with aβ-amino acid residue or a (D)-α-amino acid.
 19. A pharmaceuticalcomposition for treating hypoparathyroidism, the composition comprisinga parathyroid hormone receptor agonist-effective amount of PTH, aparathyroid hormone receptor (PTHR-1, PTHR-2) agonist-effective fragmentof PTH, a parathyroid hormone related protein (PTHrP), a PTHR-1 orPTHR-2 agonist-effective fragment of PTHrP, M-PTH, a PTHR-1 or PTHR-2agonist-effective fragment of M-PTH, BA058, or a PTHR-1 or PTHR-2agonist-effective fragment of BA058, in which at least one naturallyoccurring (L)-α-amino acid residue at position 6, 7, or 8 from theN-terminus is replaced with a β-amino acid residue or a (D)-α-aminoacid; and pharmaceutically suitable salts thereof, in combination with apharmaceutically suitable carrier.
 20. A method of treatinghypoparathyroidism in a mammalian subject, including a human subject,the method comprising administering to the subject a parathyroid hormonereceptor agonist-effective amount of a pharmaceutical composition asrecited in claim 19.